Reduced water permeability and altered ultrastructure in thin descending limb of Henle in aquaporin-1 null mice.
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It has been controversial whether high water permeability in the thin descending limb of Henle (TDLH) is required for formation of a concentrated urine by the kidney. Freeze-fracture electron microscopy (FFEM) of rat TDLH has shown an exceptionally high density of intramembrane particles (IMPs), which were proposed to consist of tetramers of aquaporin-1 (AQP1) water channels. In this study, transepithelial osmotic water permeability (Pf) was measured in isolated perfused segments (0.5-1 mm) of TDLH in wild-type (+/+), AQP1 heterozygous (+/-), and AQP1 null (-/-) mice. Pf was measured at 37 degrees C using a 100 mM bath-to-lumen osmotic gradient of raffinose, and fluorescein isothiocyanate (FITC)-dextran as the luminal volume marker. Pf was (in cm/s): 0.26 +/- 0.02 ([+/+]; SE, n = 9 tubules), 0.21 +/- 0.01 ([+/-]; n = 12), and 0.031 +/- 0.007 ([-/-]; n = 6) (P < 0.02, [+/+] vs. [+/-]; P < 0.0001, [+/+] vs. [-/-]). FFEM of kidney medulla showed remarkably fewer IMPs in TDLH from (-/-) vs. (+/+) and (+/-) mice. IMP densities were (in microm-2, SD, 5-12 micrographs): 5,880 +/- 238 (+/+); 5,780 +/- 450 (+/-); and 877 +/- 420 (-/-). IMP size distribution analysis revealed mean IMP diameters of 8.4 nm ([+/+] and [+/-]) and 5.2 nm ([-/-]). These results demonstrate that AQP1 is the principal water channel in TDLH and support the view that osmotic equilibration along TDLH by water transport plays a key role in the renal countercurrent concentrating mechanism. The similar Pf and AQP1 expression in TDLH of (+/+) and (+/-) mice was an unexpected finding that probably accounts for the unimpaired urinary concentrating ability in (+/-) mice. (+info)
Second messenger production in avian medullary nephron segments in response to peptide hormones.
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We examined the sites of peptide hormone activation within medullary nephron segments of the house sparrow (Passer domesticus) kidney by measuring rates of hormone-induced generation of cyclic nucleotide second messenger. Thin descending limbs, thick ascending limbs, and collecting ducts had baseline activity of adenylyl cyclase that resulted in cAMP accumulation of 207 +/- 56, 147 +/- 31, and 151 +/- 41 fmol. mm-1. 30 min-1, respectively. In all segments, this activity increased 10- to 20-fold in response to forskolin. Activity of adenylyl cyclase in the thin descending limb was stimulated approximately twofold by parathyroid hormone (PTH) but not by any of the other hormones tested [arginine vasotocin (AVT), glucagon, atrial natriuretic peptide (ANP), or isoproterenol, each at 10(-6) M]. Thick ascending limb was stimulated two- to threefold by both AVT and PTH; however, glucagon and isoproterenol had no effect, and ANP stimulated neither cAMP nor cGMP accumulation. Adenylyl cyclase activity in the collecting duct was stimulated fourfold by AVT but not by the other hormones; likewise, ANP did not stimulate cGMP accumulation in this segment. These data support a tubular action of AVT and PTH in the avian renal medulla. (+info)
Isoforms of the Na-K-2Cl cotransporter in murine TAL I. Molecular characterization and intrarenal localization.
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We have identified several alternatively spliced cDNAs encoding mBSC1, an apical bumetanide-sensitive Na+-K+-2Cl- cotransporter from mouse kidney. Two full-length clones were isolated, designated C4 and C9, predicting proteins of 770 and 1,095 amino acids, respectively. The C4 isoforms are generated by utilization of an alternative polyadenylation site located within the intron between exons 16 and 17 of the mBSC1 gene on chromosome 2; the resultant transcripts predict a truncated COOH terminus ending in a unique 55 amino acid sequence. The predicted C4 and C9 COOH termini differ in the distribution of putative phosphorylation sites for both protein kinase A and C. Independent splicing events involve three previously described cassette exons, which are predicted to encode most of the second transmembrane domain. A total of six different isoforms are expressed, generated by the combinatorial association of three cassette exons and two alternative 3' ends. C9-specific and C4-specific antibodies detect proteins of approximately 150 and 120 kDa, respectively, in mouse kidney. Immunofluorescence and immunohistochemistry indicate expression of both COOH-terminal isoforms within the thick ascending limb of the loop of Henle (TAL). However, staining with the C4 antibody is more heterogeneous, with a decreased proportion of positive cells in the cortical TAL. Functional expression in Xenopus oocytes indicates a dominant negative function for C4 isoforms [companion study, C. Plata, D. B. Mount, V. Rubio, S. C. Hebert, and G. Gamba. Am. J. Physiol. 276 (Renal Physiol. 45): F347-F358, 1999], and the differential expression of these isoforms may contribute to functional heterogeneity of Na+-K+-2Cl- cotransport in mouse TAL. (+info)
Isoforms of the Na-K-2Cl cotransporter in murine TAL II. Functional characterization and activation by cAMP.
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The functional properties of alternatively spliced isoforms of the mouse apical Na+-K+-2Cl- cotransporter (mBSC1) were examined, using expression in Xenopus oocytes and measurement of 22Na+ or 86Rb+ uptake. A total of six isoforms, generated by the combinatorial association of three 5' exon cassettes (A, B, and F) with two alternative 3' ends, are expressed in mouse thick ascending limb (TAL) [see companion article, D. B. Mount, A. Baekgaard, A. E. Hall, C. Plata, J. Xu, D. R. Beier, G. Gamba, and S. C. Hebert. Am. J. Physiol. 276 (Renal Physiol. 45): F347-F358, 1999]. The two 3' ends predict COOH-terminal cytoplasmic domains of 129 amino acids (the C4 COOH terminus) and 457 amino acids (the C9 terminus). The three C9 isoforms (mBSC1-A9/F9/B9) all express Na+-K+-2Cl- cotransport activity, whereas C4 isoforms are nonfunctional in Xenopus oocytes. Activation or inhibition of protein kinase A (PKA) does not affect the activity of the C9 isoforms. The coinjection of mBSC1-A4 with mBSC1-F9 reduces tracer uptake, compared with mBSC1-F9 alone, an effect of C4 isoforms that is partially reversed by the addition of cAMP-IBMX to the uptake medium. The inhibitory effect of C4 isoforms is a dose-dependent function of the alternatively spliced COOH terminus. Isoforms with a C4 COOH terminus thus exert a dominant negative effect on Na+-K+-2Cl- cotransport, a property that is reversed by the activation of PKA. This interaction between coexpressed COOH-terminal isoforms of mBSC1 may account for the regulation of Na+-K+-2Cl- cotransport in the mouse TAL by hormones that generate cAMP. (+info)
Resetting of exaggerated tubuloglomerular feedback activity in acutely volume-expanded young SHR.
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One purpose of the present study was to evaluate the ability of 7-wk-old spontaneously hypertensive rats (SHR) to reset tubuloglomerular feedback (TGF) activity in response to acute volume expansion (VE). Second, we evaluated the contribution of ANG II, via its action on AT1 receptors, to TGF control of glomerular function during VE. TGF was assessed by micropuncture methods and proximal tubular stop-flow pressure (SFP) determinations in SHR, Wistar-Kyoto rats (WKY), and Sprague-Dawley rats (SD). During euvolemia SHR exhibited enhanced TGF activity. In the same animals acute VE was achieved by infusion of saline (5 ml. h-1. 100 g body wt-1). VE led to resetting of TGF in all three strains. Maximal SFP responses, elicited by a 30-40 nl/min loop of Henle perfusion rate, decreased from 19 to 12 mmHg in SHR and, on average, from 11 to 5 mmHg in WKY and SD (P < 0.001). Tubular flow rate producing a half-maximal response (turning point) shifted to higher flow rates during VE, from 12 to 14 nl/min in SHR and from 15 to 19 nl/min in WKY. Administration of the AT1 receptor blocker candesartan (0.05 mg/kg iv) during sustained VE decreased TGF-mediated reductions in SFP in SHR and slightly increased the turning point in WKY. Nevertheless, other parameters of TGF activity were unaffected by AT1 receptor blockade. In conclusion, young SHR possess the ability to reset TGF activity in response to VE to a degree similar to compensatory adjustments in WKY. However, TGF remains enhanced in SHR during VE. ANG II and its action on AT1 receptors are in part responsible for the exaggerated SFP responses in young SHR during VE. (+info)
Nerve growth factor inhibits HCO3- absorption in renal thick ascending limb through inhibition of basolateral membrane Na+/H+ exchange.
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Nerve growth factor (NGF) inhibits transepithelial HCO3- absorption in the rat medullary thick ascending limb (MTAL). To investigate the mechanism of this inhibition, MTALs were perfused in vitro in Na+-free solutions, and apical and basolateral membrane Na+/H+ exchange activities were determined from rates of pHi recovery after lumen or bath Na+ addition. NGF (0.7 nM in the bath) had no effect on apical Na+/H+ exchange activity, but inhibited basolateral Na+/H+ exchange activity by 50%. Inhibition of basolateral Na+/H+ exchange activity with ethylisopropyl amiloride (EIPA) secondarily reduces apical Na+/H+ exchange activity and HCO3- absorption in the MTAL (Good, D. W., George, T., and Watts, B. A., III (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 12525-12529). To determine whether a similar mechanism could explain inhibition of HCO3- absorption by NGF, apical Na+/H+ exchange activity was assessed in physiological solutions (146 mM Na+) by measurement of the initial rate of cell acidification after lumen EIPA addition. Under these conditions, in which basolateral Na+/H+ exchange activity is present, NGF inhibited apical Na+/H+ exchange activity. Inhibition of HCO3- absorption by NGF was eliminated in the presence of bath EIPA or in the absence of bath Na+. Also, NGF blocked inhibition of HCO3- absorption by bath EIPA. We conclude that NGF inhibits basolateral Na+/H+ exchange activity in the MTAL, an effect opposite from the stimulation of Na+/H+ exchange by growth factors in other systems. NGF inhibits transepithelial HCO3- absorption through inhibition of basolateral Na+/H+ exchange, most likely as the result of functional coupling in which primary inhibition of basolateral Na+/H+ exchange activity results secondarily in inhibition of apical Na+/H+ exchange activity. These findings establish a role for basolateral Na+/H+ exchange in the regulation of renal tubule HCO3- absorption. (+info)
CCAAT/enhancer binding protein-beta trans-activates murine nitric oxide synthase 2 gene in an MTAL cell line.
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Nitric oxide production by nitric oxide synthase 2 (NOS2) has been implicated in epithelial cell injury from oxidative and immunologic stress. The NOS2 gene is transcriptionally activated by lipopolysaccharide (LPS) and cytokines in medullary thick ascending limb of Henle's loop (MTAL) cells and other cell types. The 5'-flanking region of the NOS2 gene contains a consensus element for CCAAT/enhancer binding proteins (C/EBP) at -150 to -142 that we hypothesized contributes to NOS2 trans-activation in the mouse MTAL cell line ST-1. Gel shift assays demonstrated LPS + interferon-gamma (IFN-gamma) induction of C/EBP family protein-DNA complexes in nuclei harvested from the cells. Supershift assays revealed that the complexes were comprised of C/EBPbeta, but not C/EBPalpha, C/EBPdelta, or C/EBPepsilon. NOS2 promoter-luciferase genes harboring deletion or mutation of the C/EBP box exhibited lower activities in response to LPS + IFN-gamma compared with wild-type NOS2 promoter constructs. Overexpression of a C/EBP-specific dominant-negative mutant limited LPS + IFN-gamma activation of the NOS2 promoter. In trans-activation assays, overexpression of C/EBPbeta stimulated basal NOS2 promoter activity. Thus C/EBPbeta appears to be an important trans-activator of the NOS2 gene in the MTAL. (+info)
Nitric oxide inhibits transcription of the Na+-K+-ATPase alpha1-subunit gene in an MTAL cell line.
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Nitric oxide (NO) has been implicated as an autocrine modulator of active sodium transport. To determine whether tonic exposure to NO influences active sodium transport in epithelial cells, we established transfected medullary thick ascending limb of Henle (MTAL) cell lines that overexpressed NO synthase-2 (NOS2) and analyzed the effects of deficient or continuous NO production [with or without NG-nitro-L-arginine methyl ester (L-NAME) in the culture medium, respectively] on Na+-K+-ATPase function and expression. The NOS2-transfected cells exhibited high-level NOS2 expression and NO generation, which did not affect cell viability or cloning efficiency. NOS2-transfected cells were grown in the presence of vehicle, NG-nitro-D-arginine methyl ester (D-NAME), or L-NAME for 16 h, after which 86Rb+ uptake assays, Northern analysis, or nuclear run-on transcription assays were performed. The NOS2-transfected cells allowed to produce NO continuously (vehicle or D-NAME) exhibited lower rates of ouabain-sensitive 86Rb+ uptake ( approximately 65%), lower levels of Na+-K+-ATPase alpha1-subunit mRNA ( approximately 60%), and reduced rates of de novo Na+-K+-ATPase alpha1-subunit transcription compared with L-NAME-treated cells. These results have uncovered a novel effect of NO to inhibit transcription of the Na+-K+-ATPase alpha1-subunit gene. (+info)